Effect of isoflavone agonists of IRF-3 on response to RNA viruses
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ABSTRACT: There is a growing need for novel antiviral therapies that are broad-spectrum, effective, and not subject to resistance due to viral mutations. Using high-throughput screening methods, including computational docking studies and an ISG54-luciferase reporter assay, we identified a class of isoflavone compounds that act as specific agonists of innate immune signaling pathways and cause activation of the IRF-3 transcription factor. The objective of the microarray study was to examine the biological pathways associated with global gene expression changes following agonist treatment. Total RNA isolation and mRNA amplification were performed on equal masses of total RNA from MRC5 cells treated with either DMSO (negative control; n=3), or 10μM of the isoflavone agonist KIN 101 (n=3) at 20 hours post treatment. As a positive control for response to an RNA virus, total RNA isolation and mRNA amplification was performed on equal masses of total RNA from MRC5 cells infected with Sendai virus (n=3) at 20 hours post infection.
Project description:Identification of genomic anchors across the MHC in untreated MRC5 fibroblasts, and fibroblasts treated with IFN-gamma, using high resolution microarrays. Identification of genomic anchors using MRC5 fibroblasts, and fibroblasts treated with IFN-gamma: Loop associated DNA vs Matrix associated DNA, 2 biological replicates
Project description:We used parkin âoverexpressing MRC5 fibroblasts to investigate the role of mitochondria deficiency on senescence-associated gene expression. RNA-seq analysis on proliferating and senescent Parkin-expressing MRC5 fibroblasts treated with CCCP (treated) or DMSO (Untreated).
Project description:The aim of this experiment was to investigate the gene expression profile of two xenografted nasopharyngeal carcinoma tumors - C15 and C17 which are international reference tools for biological investigations of this type of disease. C15 and C17 transcriptome was profiled by comparison with human untransformed diploid fibroblasts, MRC5. These fibroblasts are very sensitive to contact inhibition and were collected after 3 days of confluence, therefore in a quiescent state. We acknowledge that malignant nasopharyngeal carcinoma cells and MRC5 cells do not belong to the same lineage (epithelial cells versus fibroblasts). However this combination was expected to highlight genes involved in proliferation of NPC cells. Competitive hybridisation of cDNA for each pair C15 and MRC5, C17 and MRC5 was performed on Agilent microarrays containing 22 000 60mer oligonucleotides related to 16 000 human transcripts. The analysis was focused mainly on genes overexpressed in both C15 and C17 by comparison with MRC5
Project description:8-oxoguanine is excised from DNA by 8-oxoguanine DNA glycosylase-1 during DNA base excision repair. We used microarrays to gain insight into the potential biological function of the excised, free 8-oxoguanine base. MRC5, human diploid fibroblasts were exposed to 8-oxoguanine and changes in gene expression compared to mock control were analyzed 6 hours after exposure. MRC5, human diploid fibroblasts were exposed to 8-oxoguanine and changes in gene expression compared to mock control were analyzed 12 hours after exposure.
Project description:The goal of this experiment was to use global gene expression profiling to understand human lung epithelial cellular responses to interferon treatment. A549 cells were treated individually with IFN-α (10 ng ml−1; Sigma I4276), IFN-β (500 U ml−1; Sigma I4151) and IFN-γ (10 ng ml−1; Sigma I32265) or mock-treated. At 12 and 24 h post-treatment, cells were harvested (n = 3 wells per condition) and total RNA was extracted and analysed by microarray. Individual well replicates were pooled for each array by combining equal masses of total RNA (n=1 array per time point for each treatment and control).
Project description:KDM7A Divergent Transcript (KDM7A-DT) is a stress-induced lncRNAs. In our previous studies, KDM7A-DT showed the most robust cellular phenotype alteration and a significant TP53-dependency upon oxidative and oncogenic stress induction. To investigate the functional roles of KDM7A-DT, we first performed overexpression experiments using fibroblasts. We found that MRC5 cells overexpressing KDM7A-DT escaped cell cycle proliferation and long-term survival ability and were associated with a distinct stress-related morphology (enlarged and flattened cells) compared to cells expressing just the vehicle control. To examine the effects of KDM7A-DT overexpression on the transcriptome, we performed a differential expression gene (DEG) analysis comparing a group of four independent biological replicates of MRC5 overexpressing KDM7A-DT to a group of samples from control cells.
Project description:Three adult individuals of the Octopus vulgaris were collected from the Southern Tyrrhenian Sea (Italy). For each animal, total RNA was isolated from the supra- (SEM), sub- (SUB) esophageal masses, optic lobes (OL) and the arms (ARM). The RNA-sequencing has been performed using Illumina technology.
Project description:To study genes involved in cellular response to Influenza A virus infection, human cells MRC5, WI-38 VA-13, A549 and HEK293FT were infected with influenza A/Puerto Rico/8/1934 (H1N1) virus. RNA-seq analysis of infected cell lines after 48 h was performed on an Illumina NextSeq 500 platform. The same mock-infected cells were used as controls. PolyA RNA-enriched fraction was used for constructing of cDNA libraries. Differential expressed genes were identified using R package DESeq2.
Project description:Human cytomegalovirus (HCMV) is a widespread virus and can establish life-long latent infection in large populations. In order to establish persistent and latent infection in healthy individuals, HCMV encodes a large array of proteins that can modulate different components and pathways of host cells. It has been reported that pUL138 encoded by UL133-UL138 polycistronic locus promotes a latent infection in primary CD34+ HPCs infected in vitro. In this study, a recombinant HCMV, namely HanUL138del, was constructed by deleting the UL138 locus of Han, a clinic HCMV strain. Then a comparative quantitative proteomic analysis of Han and HanUL138del infected MRC5 cells was performed, aiming to study the effect of pUL138 on host cell in the context of HCMV infection. Our result indicated that at the early phase of HCMV infection, innate immune response was differentially activated, while at late phase of HCMV infection, multiple host proteins were differentially expressed, between Han or HanUL138del infected cells.